JP2008520798A - pH-sensitive block copolymer and polymer micelle using the same - Google Patents

Abstract

  The present invention relates to (a) a polyethylene glycol compound (A) and (b) any one or more poly (amino acid) compounds selected from the group consisting of poly (β-amino ester) and poly (amidoamine). Alternatively, a pH-sensitive block copolymer obtained by copolymerizing the copolymer (B) of the compound and a method for producing the same are provided. The present invention also provides a polymer micelle type drug composition comprising a pH-sensitive block copolymer and a physiologically active substance that can be encapsulated in the block copolymer. The block copolymer according to the present invention is obtained by copolymerizing a biodegradable poly (β-amino ester) compound having a pH sensitivity and a hydrophilic polyethylene glycol-based compound. A micelle structure can be formed by ionization characteristics that vary depending on the medium and pH, and thus can be used as a target-directed drug carrier due to changes in the body pH.

Description

  The present invention relates to a biodegradable block copolymer for a pH-sensitive drug carrier, a production method thereof, and a polymer micelle type drug composition containing the block copolymer. Deriving a block copolymer of a poly (β-amino ester) compound that has solubility in water (degree of ionization) but cannot form micelles due to a self-assembly phenomenon, and a polyethylene glycol-based compound that exhibits hydrophilicity As a result, nano-sized polymer micelles (particles) can be formed by the self-assembly phenomenon, so that not only can it be applied as a drug carrier, Instead of poly (amidoamine), which has an amide group and has a slower biodegradation rate than poly (β-amino ester), The present invention relates to a pH-sensitive block copolymer that forms micelles that can not only perform targeted drug delivery in response to changes in pH but also have an adjustable biodegradation rate, and a method for producing the same.

  A micelle generally refers to a thermodynamically stable and uniform spherical structure formed by a low molecular weight substance having an amphiphilic property, for example, a hydrophilic group and a hydrophobic group. When an insoluble drug is dissolved and introduced into the compound having the micelle structure, the drug is present inside the micelle. Since this type of micelle can deliver a targeted drug in response to changes in temperature and pH in the body, it has a very high applicability as a drug carrier.

  Korean Patent Application No. 10-2001-0035265 describes the production of micelles using polyethylene glycol and biodegradable polymers. Since these substances are all biodegradable, there is a merit that they have bioaffinity, but they are not sensitive to changes in the body, for example, specific changes such as pH. The disadvantage is that delivery of the drug to the site is difficult.

On the other hand, the pH environment in the body usually shows pH 7.2 to 7.4, but the surrounding environment of abnormal cells such as cancer cells is known to be slightly acidic with pH 6.0 to 7.2. ing. In recent years, in order to deliver a specific drug to cancer cells, research has been conducted to deliver a drug at pH 7.2 or lower.
Korean Patent Application No. 10-2001-0035265 US Pat. No. 6,103,865

  US Pat. No. 6,103,865 shows a polymer using a sulfonamide, which is a pH-sensitive substance. However, the sulfonamide becomes insoluble at pH 7.4 or lower, and at pH 7.4 or higher. Ionized and acidic. In this case, since it shows the opposite characteristics to the target for cancer cells, it can be said that a compound having basicity is required for the target for cancer cells.

  US 2004/0071654 A1 describes the production of a poly (β-amino ester) that exhibits basicity, but the poly (β-amino ester) is an ester group in the main chain as a kind of poly (amino acid). And a tertiary amine group, and has the merit of exhibiting ionization characteristics in which solubility in water changes depending on pH.

  The present inventors pay attention to the fact that when poly (β-amino ester) or poly (amidoamine), which is a kind of poly (amino acid), is used as a simple substance, micelles cannot be formed due to the self-assembly phenomenon although it shows pH dependence. Then, when a block copolymer is formed by copolymerizing a hydrophilic polyethylene glycol compound with the poly (β-amino ester) or a mixture of poly (β-amino ester) and poly (amidoamine). It has been found that the block copolymer forms a micelle structure capable of target delivery at a specific pH, and as a result, can be applied as a carrier for sustained-release drug delivery with controlled biodegradation rate. The invention has been completed. In order to solve the problem that the function as a drug transporter cannot be normally performed due to the high decomposition rate of poly (β-amino ester), the present inventors mainly used poly (β-amino acid) as a kind of poly (β-amino acid). The chain was copolymerized with a poly (amidoamine), which is composed of an amide group instead of an ester group and has a relatively slow degradation rate, and was successfully adjusted to maintain the desired biodegradation rate.

  Therefore, the present invention provides a block copolymer obtained by polymerization of poly (β-amino ester) (PAE), poly (amidoamine) (PAA) or a copolymer thereof (PAEA) and a polyethylene glycol-based compound (MPEGA). It is an object of the present invention to provide a polymer, a production method thereof, and a polymer micelle type drug composition containing the block copolymer.

  The present invention relates to (a) a polyethylene glycol compound (A) and (b) any one or more poly (amino acid) compounds selected from the group consisting of poly (β-amino ester) and poly (amidoamine). Alternatively, a pH-sensitive block copolymer obtained by copolymerizing with the copolymer (B) of the compound and a method for producing a polymer micelle using the same are provided.

  Furthermore, the present invention provides a polymer micelle type drug composition comprising a pH-sensitive block copolymer and a physiologically active substance that can be encapsulated in the block copolymer.

  Hereinafter, the present invention will be described in detail.

  The present invention is based on the copolymerization of a pH-sensitive poly (amino acid) compound such as poly (β-amino ester), poly (amidoamine) or a copolymer thereof with a hydrophilic polyethylene glycol compound. To provide a block copolymer that not only is sensitive to changes in pH in the body, but also capable of forming a micelle structure in a specific pH range, and in which the biodegradation rate of the formed micelle in vivo is controlled. It is characterized by.

  The pH-sensitive micelle according to the present invention forms stable micelles at a specific pH, for example, pH 7.2 to 7.4, which is the pH range of normal cells in the body, and pH 7. In 2 or less, the micelle structure is disrupted, so that it can be used as a carrier for drug delivery targeted to cancer cells. That is, at low pH (pH 7.0 or less), it exists in poly (amino acid) poly (β-amino ester) (PAE), poly (amidoamine) (PAA) or a copolymer thereof (PAEA) 3 Since the degree of ionization of the primary amine is increased, the whole PAE (or PAA, PAEA) changes to water solubility and cannot form micelles. At pH 7.4, the degree of ionization of PAE (or PAA, PAEA) decreases. By exhibiting hydrophobicity, micelles are formed by self-assembly.

  In addition, the block copolymer capable of forming a pH-sensitive micelle is used not only in the fields of gene delivery and drug delivery, but also by delivering a substance for diagnosing a disease to a non-normal cell, thereby enabling diagnosis such as diagnostic imaging. It can also be applied to applications.

  In addition, in the present invention, micelles are formed in the same pH range of 7.2 to 7.4 as normal body conditions, and the micelles are disintegrated in pH 7.2 or lower, which is an abnormal condition such as cancer cells. Such cancer cell target-oriented micelles have been devised and applied, but by appropriately changing the components of the block copolymer, their molar ratio, molecular weight and / or functional groups in the block, cancer cells In addition, it can be applied effectively by devising target-oriented micelles for gene mutation or other application fields.

  Furthermore, in the present invention, the formation conditions of the pH-sensitive block copolymer, for example, the constituent components of the block copolymer, their molar ratio, molecular weight and / or functional group in the block are variously adjusted. The biodegradation rate of the pH-sensitive block copolymer micelles in vivo can be easily adjusted, thereby delivering the drug in a targeted manner to the appropriate place in the body where the drug delivery is to be performed. Can do.

  As one of the constituent components of the block copolymer that forms a pH-sensitive micelle according to the present invention, a biodegradable compound having hydrophilicity well known in the art can be used without limitation. These compounds can be suitably used. More preferably, it has a single functional group such as acrylate or methacrylate at the end of the polyethylene glycol-based compound, and as an example, the following general formula 1:

[式中、Ｒは、水素原子若しくは炭素原子数１〜６のアルキル基であり、このとき、ｘは、１〜１０，０００の範囲の自然数である]
の化合物がある。

[Wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and x is a natural number in the range of 1 to 10,000]
There are compounds.

  The alkyl group means a linear or branched lower saturated aliphatic hydrocarbon such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, and the like. There are n-pentyl groups and the like.

  Although there is no restriction | limiting in particular in the molecular weight (Mn) of the said polyethyleneglycol type compound, It is preferable that it is the range of 500-5000. When the molecular weight (Mn) of the polyethylene glycol-based compound is outside the above range, for example, when it is less than 500 or exceeds 5,000, the molecular weight of the finally obtained block copolymer is only difficult to adjust. In addition, it is not easy to form micelles using the block copolymer. That is, when the molecular weight is less than 500, the hydrophilic block at a specific pH is too short, and self-assembly due to hydrophilicity / hydrophobicity does not occur. As a result, it is difficult to form micelles, and even micelles are formed. Even so, it is easily dissolved and disintegrated in water. Also, if the molecular weight exceeds 5,000, the block is too long compared to the molecular weight of the hydrophobic poly (amino acid) and the hydrophilic / hydrophobic balance is lost, so that micelles cannot be formed at a specific pH and precipitate. There are things to do.

  Another component of the block copolymer that forms pH-sensitive micelles according to the present invention is a poly (amino acid) compound having both hydrophobicity and pH sensitivity. Non-limiting examples include poly (β-amino ester) (PAE), poly (amidoamine) (PAA), or a mixed copolymer thereof (PAEA).

  As described above, the PAE, PAA, and PAEA, which are poly (amino acids), have ionization characteristics in which the solubility in water varies depending on pH depending on the tertiary amine group present in the body. In response to changes, micelle structures can be formed and / or collapsed. The compound can be produced by a well-known method in the art. For example, an amine compound is polymerized to a bisacrylate compound or a bisacrylamide compound having a double bond through a Michael reaction to obtain a polymer. An (amino acid) compound can be obtained.

  The bisacrylate compound used at this time is represented by the following formula 2:

[式中、Ｒ_３は、炭素原子数１〜３０のアルキル基である]
で表わすことができ、この非制限的な例としては、エチレングリコールジアクリレート、１，４−ブタンジオールジアクリレート、１，３−ブタンジオールジアクリレート、１，６−ヘキサンジオールジアクリレート、１，６−ヘキサンジオールエトキシレートジアクリレート、１，６−ヘキサンジオールプロポキシレートジアクリレート、３−ヒドロキシ−２，２−ジメチルプロピル３−ヒドロキシ−２，２−ジメチルプロピオネートジアクリレート、１，７−ヘプタンジオールジアクリレート、１，８−オクタンジオールジアクリレート、１，９−ノナンジオールジアクリレート、１，１０−デカンジオールジアクリレート若しくはこれらの混合物などがある。

[Wherein R ₃ is an alkyl group having 1 to 30 carbon atoms]
Non-limiting examples of this include ethylene glycol diacrylate, 1,4-butanediol diacrylate, 1,3-butanediol diacrylate, 1,6-hexanediol diacrylate, 1,6 -Hexanediol ethoxylate diacrylate, 1,6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, 1,7-heptanediol Examples include diacrylate, 1,8-octanediol diacrylate, 1,9-nonanediol diacrylate, 1,10-decanediol diacrylate, or a mixture thereof.

  Bisacrylamide compounds are represented by the following general formula 3:

[式中、Ｒは、炭素原子数１〜２０のアルキル基である]
で表わすことができる。このとき、ジアクリルアミドの非制限的な例としては、Ｎ，Ｎ’−メチレンビスアクリルアミド（ＭＤＡ）、Ｎ，Ｎ’−エチレンビスアクリルアミド若しくはこれらの混合物などがある。前記ビスアクリルアミド系の化合物と、アミン系の化合物、例えば４−アミノメチルピペリジン（ＡＭＰＤ）、Ｎ−メチルエチレンジアミン（ＭＥＤＡ）、または１−（２−アミノエチル）ピペリジン（ＡＥＰＺ）とを、当業界における通常の方法、例えばミカエル反応により反応させ、ポリ（アミノ酸）を製造することが可能である。

[Wherein R is an alkyl group having 1 to 20 carbon atoms]
It can be expressed as At this time, non-limiting examples of diacrylamide include N, N′-methylenebisacrylamide (MDA), N, N′-ethylenebisacrylamide or a mixture thereof. The bisacrylamide compound and an amine compound such as 4-aminomethylpiperidine (AMPD), N-methylethylenediamine (MEDA), or 1- (2-aminoethyl) piperidine (AEPZ) in the art. It is possible to produce poly (amino acid) by reacting by an ordinary method, for example, Michael reaction.

  In addition, the amine compound can be used without limitation as long as it has an amine group, and in particular, the following general formula 4:

で表わされる１級アミン、下記一般式５：

A primary amine represented by the following general formula 5:

[式中、Ｒ_１及びＲ_２は、炭素原子数１〜２０のアルキル基である]
で表わされる２級アミン含有のジアミン化合物若しくはこれらの混合物などが好適に使用可能である。

[Wherein R ₁ and R ₂ are alkyl groups having 1 to 20 carbon atoms]
A secondary amine-containing diamine compound represented by the above or a mixture thereof can be suitably used.

  Non-limiting examples of the primary amine compound include 3-methyl-4- (3-methylphenyl) piperazine, 4- (ethoxycarbonylmethyl) piperazine, 4- (phenylmethyl) piperazine, 4- (1- Phenylethyl) piperazine, 4- (1,1-dimethoxycarbonyl) piperazine, 4- (2- (bis- (2-propenyl) amino) ethyl) piperazine, methylamine, ethylamine, butylamine, hexylamine, 2-ethylhexylamine 2-piperidin-1-ethylamine, C-aziridin-1-yl-methylamine, 1- (2-aminoethyl) piperazine, 4- (aminomethyl) piperazine, N-methylethylenediamine, N-ethylethylenediamine, N- Hexylethylenediamine, picolamine, adenine Non-limiting examples of the secondary amine-containing diamine compounds include piperazine, piperidine, pyrrolidine, 3,3-dimethylpiperidine, 4,4′-trimethylenedipiperidine, N, N′-dimethyl. Examples include ethylenediamine, N, N′-diethylethylenediamine, imidazolidine or diazepine.

  In the production of poly (amino acids) exhibiting pH sensitivity, such as PAE, PAA, and PAEA, the reaction molar ratio of the bisacrylate compound or bisacrylamide compound to the amine compound is 1: 0.5 to 2.0. It is preferable that it is the range of these. When the molar ratio of the amine compound is less than 0.5 or exceeds 2.0, the molecular weight of the polymer to be polymerized is 1000 or less, so that it becomes difficult to form micelles.

  The pH-sensitive block copolymer according to the present invention obtained by copolymerization of a hydrophilic polyethylene glycol-based compound as described above and poly (amino acid) has the following general formula 6, 7, or 8:

[式中、
Ｒは、水素原子若しくは炭素原子数１〜６のアルキル基であり、このとき、ｘは、１〜１０，０００の範囲の自然数であり、
Ｒ_１及びＲ_２は、炭素原子数１〜２０のアルキル基であり、
Ｒ_３は、炭素原子数１〜３０のアルキル基であり、
Ｒ_４は、炭素原子数１〜２０のアルキル基であり、
ｙは、１〜１０，０００の範囲の自然数である]
で表わすことができる。

[Where
R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, wherein x is a natural number in the range of 1 to 10,000,
R ₁ and R ₂ are alkyl groups having 1 to 20 carbon atoms,
R ₃ is an alkyl group having 1 to 30 carbon atoms,
R ₄ is an alkyl group having 1 to 20 carbon atoms,
y is a natural number in the range of 1 to 10,000]
It can be expressed as

  The block copolymer represented by the general formula 6, 7 or 8 can form micelles or collapse depending on the pH change due to the above-mentioned amphiphilicity and pH sensitivity, and preferably has a pH of 7.0 to 7.0. When it is in the range of 7.4, micelles are formed, and when the pH is in the range of 6.5 to 7.0, the micelles are disintegrated. In particular, the block copolymer according to the present invention has the merit that it exhibits excellent sensitivity within the range of pH ± 0.2, and therefore, for applications where sensitivity due to changes in the body pH is required, for example, a carrier for drug delivery or Satisfactory results can be derived in diagnostic applications.

  As long as the block copolymer according to the present invention maintains pH sensitivity and maintains micelle-forming physical properties, in addition to the above-mentioned hydrophilic polyethylene glycol-based compound and poly (amino acid) compound, it is a common unit in the industry. The body may further be included, and this also belongs to the category of the present invention.

  Although there is no restriction | limiting in particular in the molecular weight range of the said block copolymer, It is preferable that it is the range of 1,000-20,000. When the molecular weight is less than 1,000, not only is it difficult to form block copolymer micelles at a specific pH, but even if micelles are formed, they are easily dissolved and disintegrated in water. On the other hand, when the molecular weight exceeds 20,000, the hydrophilic / hydrophobic balance may be lost, and micelles may not be formed at a specific pH and may precipitate.

  The content of the polyethylene glycol block (A) in the pH-sensitive block copolymer according to the present invention is not particularly limited, but is preferably 5 to 95 parts by weight, and preferably 10 to 40 parts by weight. Part. When the content of the polyethylene glycol block is less than 5 parts by weight, the block copolymer may precipitate without forming micelles. When the content exceeds 95 parts by weight, the blocks forming the interior of the micelles are too small. Thus, micelles cannot be formed and remain dissolved. Further, the block copolymer may be prepared by adjusting a reaction molar ratio of a polyethylene glycol-based compound and a poly (amino acid), for example, PAE, PAA, or PAEA, so that an AB type diblock copolymer, ABA or A BAB-type triblock copolymer or a variety of other blocks can be formed.

  The pH-sensitive block copolymer according to the present invention can be produced according to a usual method in the art, and in this example, it can be synthesized through the following reaction formula 1, 2 or 3.

  An example of the production method represented by the reaction formula 1 is as follows. A well-known copolymerization reaction is carried out by those skilled in the art using polyethylene glycol (MPEG-A), primary amine and bisacrylate having an acrylate end group. At this time, the primary amine and bisacrylate form a poly (β-amino ester) by Michael addition reaction, and the formed poly (β-amino ester) is a polyethylene glycol-based compound having an acrylate functional group at the terminal. The block copolymer represented by the general formula 6 can be obtained.

  One example of the production method represented by the reaction formula 2 is represented by the general formula 7 using a polyethylene glycol (MPEG-A) having an acrylate end group, a diamine compound containing a secondary amine, and a bisacrylate. A block copolymer can be obtained, and chloroform, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, methylene chloride, and the like can be used as the organic solvent in the production of the block copolymer.

  One example of the production method represented by the above reaction scheme 3 is a well-known copolymerization by those skilled in the art using polyethylene glycol having an acrylate end group (MPEG-A), primary or secondary amine, and bisacrylamide. Perform the reaction. At this time, the primary or secondary amine and bisacrylate form a poly (amino acid) called poly (amidoamine) by Michael addition reaction, and the formed poly (amidoamine) is converted into a polyethylene glycol system having an acrylate functional group at the terminal. The pH-sensitive block copolymer represented by the general formula 8 can be obtained by copolymerization with the above compound.

  On the other hand, in the present invention, gel permeation chromatography (GPC) is used to measure the molecular weight of the block copolymer synthesized in this way, and the micelle concentration change and micelle size change due to pH change. In order to measure the change, a fluorescence spectrometer and a particle size measuring device (DLS: Dynamic Light Scattering) were used. In fact, the applicability as a pH-sensitive micelle could be confirmed by the analysis as described above.

  The present invention also relates to a polymer micelle-type drug comprising (a) the aforementioned block copolymer that forms micelles in response to pH changes, and (b) a physiologically active substance that can be encapsulated in the block copolymer. A composition is provided.

  The polymeric micelle-type drug composition forms micelles when taken into the body, and is encapsulated by the collapse of the micelles when a low pH location such as cancer cells is reached locally. Targeted drug delivery can be achieved by release of the supported drug.

  There are no particular restrictions on the physiologically active substance that can be encapsulated in the polymeric micelle block copolymer according to the present invention. Non-limiting examples include anticancer agents, antibacterial agents, steroids, anti-inflammatory analgesics, sex There are hormones, immunosuppressants, antiviral agents, anesthetics, antiemetics, or antihistamines. In addition to the above-mentioned components, it can also contain conventional additives in the industry such as excipients, stabilizers, pH adjusters, antioxidants, preservatives, binders, or disintegrants. .

  The polymer micelle production method according to the present invention can be used alone or in combination with methods such as stirring, heating, ultrasonic irradiation, solvent evaporation using an emulsification method, matrix formation or dialysis using an organic solvent. .

  Although there is no restriction | limiting in particular in the diameter of the obtained polymer micelle, It is preferable that it is the range of 10-1000 nm. The polymeric micelle drug composition can be formulated and used as an oral or parenteral preparation, and can be produced as an intravenous, intramuscular or subcutaneous injection.

  Furthermore, the present invention provides a method of using the pH-sensitive block copolymer as a carrier for drug transport or disease diagnosis. At this time, the substance contained in the block copolymer is not particularly limited as long as it is a substance for treating, preventing or diagnosing diseases.

  In addition to these, the present invention provides (a) any one or more compounds selected from the group consisting of (a) a compound containing an ester group and a tertiary amine group, and a compound containing an amide group and a tertiary amine group, Provided is a method for producing a pH-sensitive block copolymer capable of forming micelles by copolymerizing a compound copolymer and (b) a hydrophilic or amphiphilic compound.

[Best Mode for Carrying Out the Invention]
Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, the following examples are merely for illustrating the present invention, and the scope of the present invention is not limited thereto.

[Examples 1 to 20]
Synthesis of pH-sensitive block copolymer [Example 1]
(Production of polyethylene glycol-poly (β-amino ester) block copolymer (PAE))
Polyethylene glycol methyl ether (MPEG2000, Mn = 2000) and acryloyl chloride are reacted in the presence of methylene chloride containing triethylamine (TEA), extracted with dilute hydrochloric acid aqueous solution, and precipitated into n-hexane, which is a hydrophilic component. Polyethylene glycol methyl ether acrylate (MPEG2000-A, Mn = 2000) was produced. MPEG2000-A 0.1 mol, 4,4′-trimethylenedipiperidine 1 mol and 1,6-hexanediol diacrylate 1 mol as a diamine component were put in a two-necked round flask and, after decompression, filled with nitrogen. . At this time, chloroform was used as a reaction solvent and reacted at 50 ° C. for 48 hours to produce a polyethylene glycol-poly (β-amino ester) block copolymer (MPEGA-PAE) having a molecular weight (Mp) of 7700. .

[Example 2]
A molecular weight (Mp) of 4800 was obtained in the same manner as in Example 1 except that the amount of polyethylene glycol methyl ether acrylate (MPEG2000-A) was changed from 0.1 mol to 0.2 mol. An MPEGA-PAE block copolymer was produced.

[Example 3]
A molecular weight (Mp) of 4400 was obtained in the same manner as in Example 1 except that the amount of polyethylene glycol methyl ether acrylate (MPEG2000-A) was changed from 0.1 mol to 0.3 mol. An MPEGA-PAE block copolymer was produced.

[Example 4]
An MPEGA-PAE block having a molecular weight (Mp) of 13500 is the same as the method of Example 1 except that instead of MPEG2000, 0.1 mol of MPEG5000-A manufactured using MPEG5000 is used. A polymer was produced.

[Example 5]
An MPEGA-PAE block having a molecular weight (Mp) of 12500 was used in the same manner as in Example 1 except that 0.4 mol of MPEG5000-A manufactured using MPEG5000 was used instead of MPEG2000. A polymer was produced.

[Example 6]
Example 1 except that, instead of MPEG2000, 0.1 mol of MPEG5000-A prepared using MPEG5000 was used and 0.8 mol of 4,4′-trimethylenedipiperidine was used. In the same manner as described above, an MPEGA-PAE block copolymer having a molecular weight (Mp) of 8800 was produced.

[Example 7]
Example 1 except that, instead of MPEG2000, 0.1 mol of MPEG5000-A prepared using MPEG5000 was used and 1.1 mol of 4,4′-trimethylenedipiperidine was used. The MPEGA-PAE block copolymer having a molecular weight (Mp) of 20000 was produced in the same manner as in the above method.

[Example 8]
Example 1 except that, instead of MPEG2000, 0.1 mol of MPEG5000-A produced using MPEG5000 was used and 1.3 mol of 4,4′-trimethylenedipiperidine was used. In the same manner as described above, an MPEGA-PAE block copolymer having a molecular weight (Mp) of 7900 was produced.

[Example 9]
Example 1 except that, instead of MPEG2000, 0.1 mol of MPEG5000-A prepared using MPEG5000 was used and 1.5 mol of 4,4′-trimethylenedipiperidine was used. In the same manner as described above, an MPEGA-PAE block copolymer having a molecular weight (Mp) of 6900 was produced.

[Example 10]
Example 1 except that MPEG5000-A 0.1 mol produced using MPEG5000 was used instead of MPEG2000, and piperazine was used instead of 4,4'-trimethylenedipiperidine. In the same manner as in Method 1, an MPEGA-PAE block copolymer having a molecular weight (Mp) of 6200 was produced.

[Example 11]
In order to adjust the biodegradation rate of the MPEG-PAE block copolymer, 0.1 mol of MPEG5000-A produced using MPEG5000 was used, and 1 mol of piperazine as an amine compound and 1,6 -Add 0.2 mol of N, N'-methylenebisacrylamide as a diacrylamide component, which is a biodegradation rate regulator, to 0.8 mol of hexanediol diacrylate, and add MPEGA-PAEA block of molecular weight 13,200. A polymer was produced.

[Example 12]
Example 11 except that 0.4 mol of N, N′-methylenebisacrylamide as a diacrylamide component as a biodegradation rate regulator was added to 0.6 mol of 1,6-hexanediol diacrylate. In the same manner as described above, an MPEGA-PAEA block copolymer having a molecular weight (Mp) of 13,700 was produced.

[Example 13]
Example 11 except that 0.6 mol of N, N′-methylenebisacrylamide as a diacrylamide component as a biodegradation rate regulator was added to 0.4 mol of 1,6-hexanediol diacrylate. In the same manner as described above, an MPEGA-PAEA block copolymer having a molecular weight (Mp) of 13,400 was produced.

[Example 14]
Example 11 except that 0.8 mol of N, N′-methylenebisacrylamide as a diacrylamide component as a biodegradation rate regulator was added to 0.2 mol of 1,6-hexanediol diacrylate. In the same manner as described above, an MPEGA-PAEA block copolymer having a molecular weight (Mp) of 13,300 was produced.

[Example 15]
Using 0.1 mole of MPEG5000-A produced using MPEG5000, 1 mole of piperazine as an amine compound and 1 mole of N, N′-methylenebisacrylamide as a diacrylamide compound were added in each mole. , 800 MPEGA-PAA block copolymers were produced.

[Example 16]
An MPEGA having a molecular weight of 10,200 was used in the same manner as in Example 11 except that 0.1 mol of MPEG2000-A produced using MPEG2000 was used instead of 0.1 mol of MPEG5000-A. -PAEA block copolymer was prepared.

[Example 17]
Using 0.1 mol of MPEG2000-A produced using MPEG2000, 0.6 mol of 1,6-hexanediol diacrylate was added to N, N′-methylenebis as a diacrylamide component as a biodegradation rate regulator. An MPEGA-PAEA block copolymer having a molecular weight (Mp) of 10,400 was produced in the same manner as in Example 11 except that 0.4 mol of acrylamide was added.

[Example 18]
Using 0.1 mol of MPEG2000-A produced using MPEG2000, 0.4 mol of 1,6-hexanediol diacrylate was added to N, N′-methylenebis as a diacrylamide component as a biodegradation rate regulator. An MPEGA-PAEA block copolymer having a molecular weight (Mp) of 10,700 was produced in the same manner as in Example 11 except that 0.6 mol of acrylamide was added.

[Example 19]
Using 0.1 mol of MPEG2000-A produced using MPEG2000, 0.2 mol of 1,6-hexanediol diacrylate was added to N, N′-methylenebis as a diacrylamide component as a biodegradation rate regulator. An MPEGA-PAEA block copolymer having a molecular weight (Mp) of 10,300 was produced in the same manner as in Example 16 except that 0.8 mol of acrylamide was used.

[Example 20]
Using 0.1 mole of MPEG2000-A produced using MPEG2000, 1 mole of piperazine as an amine compound and 1 mole of N, N′-methylenebisacrylamide as a diacrylamide compound are added, and the molecular weight is increased. 10,600 MPEGA-PAA block copolymers were prepared.

[Comparative Examples 1-2]
[Comparative Example 1]
An MPEGA-PAE block having a molecular weight (Mp) of 8000 was used in the same manner as in Example 1 except that instead of MPEG2000, 0.1 mol of MPEG500-A produced using MPEG400 was used. A polymer was produced.

  Using the manufactured MPEGA-PAE block copolymer, the behavior due to pH change was observed, and it was confirmed that the block copolymer could not form micelles. This is because, as described above, at a specific pH, the hydrophilic block is too short and self-assembly due to hydrophilicity / hydrophobicity does not occur. As a result, micelles are not easily formed, and even micelles are formed. Even so, it is proved that it is easily dissolved and dissolved in water.

[Comparative Example 2]
An MPEGA-PAE block having a molecular weight (Mp) of 14500 is the same as the method of Example 1 except that instead of MPEG2000, 0.1 mol of MPEG6000-A manufactured using MPEG6000 is used. A polymer was produced.

  When the behavior due to pH change was observed using the MPEGA-PAE block copolymer, it was confirmed that the block copolymer according to Comparative Example 2 could not form micelles as in Comparative Example 1. This means that the block is too long compared to the molecular weight of the poly (amino acid) which is hydrophobic and the balance between hydrophilicity / hydrophobicity is lost, and micelles cannot be formed at a specific pH and precipitate. .

[Experimental Example 1. Measurement of molecular weight of pH-sensitive block copolymer]
In order to determine the molecular weight of the pH-sensitive block copolymer prepared according to the present invention, analysis was performed as follows.

  To examine the possibility of adjusting these molecular weights using MPEGA-PAE, MPEGA-PAA or MPEGA-PAEA block copolymers prepared according to Examples 1-15, gel permeation chromatography (GPC, Waters) Analysis).

  First, an analysis of the molecular weight of the MPEGA-PAE block copolymer produced according to Examples 1 to 5 was performed, and it is possible to adjust the molecular weight of the final copolymer using MPEG2000-A. (See FIG. 1) When using MPEG5000-A, it was found that the final molecular weight adjustment of the copolymer was not easy (see FIG. 2).

  In order to investigate the possibility of adjusting the molecular weight of the final copolymer obtained from MPEG5000-A, the contents of MPEG-A and bisacrylate were kept as they were, and only the contents of diamine were changed. Analysis of the molecular weights for MPEGA-PAE block copolymers of ~ 9 showed that, even if MPEG5000-A was used, the final copolymer molecular weight was not only adjustable, but also di- It was confirmed that the final copolymer having a block or tri-block structure can be adjusted (see FIG. 3). In fact, when the amount of diamine added was 1 mol, a copolymer having a molecular weight of about 13,000 was synthesized, which was a diblock in which MPEG having a molecular weight of 5000 and PAE having a molecular weight of 8000 were combined. Presumed to be a copolymer. Further, it can be seen that when the amount of diamine added is increased to 1.1 mol, the molecular weight is increased to about 19,000, although the molecular weight should be lowered due to variation in the molar ratio during polymerization. From this, it can be predicted that a triblock copolymer composed of MPEG-A, PAE, and MPEG-A was produced. However, when the addition amount of diamine was increased to 1.3 mol and 1.5 mol, the molecular weight of the block copolymer was decreased, but this was due to the variation in molar ratio greatly affecting the degree of polymerization. It is recognized that

[Experimental Example 2. Measurement of micelle change due to pH change]
In order to observe the behavior of the pH-sensitive block copolymer micelles prepared according to the present invention due to pH change, experiments were conducted as follows.

  Various molecular weight block copolymers prepared according to Examples 1-4 and Example 10 were used. Pyrene, which is a hydrophobic luminescent material, was used because the change in behavior of micelles cannot be measured directly depending on the fluorescence analyzer.

A pH 6.0 buffer solution containing 10 ⁻⁶ M pyrene was prepared, and each copolymer prepared according to Examples 1 to 4 and Example 10 was dissolved to a concentration of 1 mg / mL, and then pH 8.0. The pH of the solution was increased. Thereafter, a 5M aqueous hydrochloric acid solution was added dropwise to change the pH to the range of 5.5 to 8.0, and then the change in emission energy due to the change in micelle concentration was measured with a fluorescence spectrometer.

  First, when the behavioral change according to pH change was measured with respect to the block copolymer of Example 1, Example 2 and Example 3 manufactured using polyethylene glycol having a molecular weight of 2000, poly (β-amino) was measured. It was found that as the molecular weight of the ester) block copolymer increases, the pH range in which the micelles collapse is slightly different (see FIG. 4). This is because the degree of ionization due to pH change is slightly different due to the change in the fraction of the hydrophobic poly (β-amino ester) block and the hydrophilic polyethylene glycol block in the produced block copolymer. It means that the behavior range of the micelle changes.

  Moreover, when the behavior of the copolymer micelles produced using different diamine compounds according to Example 4 and Example 10 was measured, the copolymer of Example 4 was more micelle in a narrower pH range. It was found that collapse occurred (see FIG. 5). This is recognized as a phenomenon caused by the difference in ionization tendency between piperazine and 4,4'-trimethylenedipiperidine. For this reason, when 4,4′-trimethylenedipiperidine is used, it can be confirmed that the micelle collapse occurs in a narrower range and can be used as a substance more sensitive to pH change. It was.

[Experimental Example 3. Measurement of micelle change due to pH of block copolymer]
In order to observe the change at a specific pH of the pH-sensitive block copolymer micelle prepared according to the present invention, an experiment was conducted as follows.

(3-1. Measurement of CMC)
CMC at pH 7.01, pH 7.24 and pH 7.4 was measured for the copolymer produced according to Example 4.

  As a result of the experiment, it was confirmed that the block copolymer according to the present invention formed stable micelles at pH 7.4, whereas no micelles were formed at pH 7.0 (see FIG. 6). ). This is because, at low pH (pH 7.0 or lower), the PAE as a whole becomes water-soluble by increasing the degree of ionization of the tertiary amine present in the poly (β-amino ester), so that the copolymer forms micelles. On the other hand, at pH 7.4, the degree of ionization of PAE decreases to show hydrophobicity, which means that micelles are formed by self-assembly.

(3-2. Measurement of micelle dimensions)
The copolymer produced according to Example 4 was measured for changes in micelle dimensions at pH 8.01, pH 7.42, pH 7.23, pH 6.68, pH 6.30 using DLS (dynamic light scattering). .

  As a result of the experiment, it was confirmed that micelles having a certain size were present at pH 7.0 or higher (see FIGS. 7, 8 and 9). However, at pH below 7.0, poly (β-amino was present. It was confirmed that the ester) was completely ionized and no micelles were formed (see FIG. 10).

  This confirms that the pH-sensitive block copolymer according to the present invention can form and collapse polymeric micelles through the amphipathicity present in the copolymer and the reversible self-assembly phenomenon due to pH change. did it.

[Experimental Example 4. Evaluation of biodegradation rate of block copolymer micelle by pH]
In order to observe the change at a specific pH of the pH-sensitive block copolymer micelle prepared according to the present invention, an experiment was conducted as follows.

  In the experiment, the copolymer of Example 4 produced using poly (β-aminoester) having an ester group in the main chain and a relatively high biodegradation rate in vivo, and an amide in the main chain The copolymer of Example 11 prepared using poly (amidoamine) having a group and a relatively slow biodegradation rate in vivo was used. In addition, the content of the components constituting the copolymer of Example 11 such as an amine compound and poly (amidoamine) as a biodegradation rate regulator, and the composition ratio thereof were obtained. The copolymers of Examples 11 to 20 were used.

  As a result of measuring the change over time in the molecular weight of each of the copolymers at pH 7.4, the block copolymer micelle according to Example 4 had a molecular weight reduced to more than half within 30 hours. It was found that the block copolymer micelle according to Example 11 had a slow biodegradation rate (see FIG. 11). Furthermore, as a result of measurement using the block copolymer micelles of Example 11 to Example 20, by adjusting the content of the components constituting the block copolymer micelles and their molar ratios, etc. in vivo It was confirmed that it was easy to adjust the micelles to show various decomposition rates (see FIG. 12). For this reason, the pH-sensitive block copolymer according to the present invention is used as a kind of poly (β-amino acid) through a copolymer with poly (amidoamine) having a relatively low biodegradation rate in vivo. It was confirmed that not only the degradation rate can be adjusted easily, but also that the biodegradation rate can be maintained continuously.

[Industrial applicability]
The block copolymer according to the present invention is a poly (amino acid), such as poly (β-amino ester) and poly (amidoamine) compounds, which have solubility in water depending on pH but cannot form micelles due to the self-assembly phenomenon In addition, by polymerizing a hydrophilic polyethylene glycol-based compound to obtain a pH-sensitive block copolymer, it is possible not only to maintain pH sensitivity but also to form polymer micelles reversibly by a self-assembly phenomenon. This allows it to be used as a drug transporter targeted for changes in body pH and for diagnostic applications.

  Although the invention has been described with reference to the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments and drawings. On the other hand, it is intended to encompass various changes and modifications within the spirit and scope of the appended claims.

FIG. 1 shows MPEG-A in the pH-sensitive block copolymer of Examples 1 to 3 produced using polyethylene glycol methyl ether acrylate (MPEG-A) having a molecular weight of 2000 and poly (β-amino ester). It is a graph which shows the adjustment result of the molecular weight of the block copolymer by the change of the molar ratio of (Mn = 2000). FIG. 2 shows the moles of MPEG-A (Mn = 5000) in the pH-sensitive block copolymers of Examples 4 and 5 prepared using MPEG-A having a molecular weight of 5000 and poly (β-amino ester). It is a graph which shows the adjustment result of the molecular weight of the block copolymer by the change of ratio. FIG. 3 is a graph showing the results of adjusting the molecular weight of the block copolymer by changing the molar ratio of the diamine in the pH-sensitive block copolymer produced according to Examples 4 and 6-9. FIG. 4 is a graph showing changes in the behavior of micelles due to changes in pH in the block copolymers produced according to Examples 1 to 3. FIG. 5 is a graph showing the change in behavior of micelles due to pH change in the block copolymers of Example 4 and Example 10 prepared using piperazine and 4,4′-trimethylenedipiperidine as diamine compounds, respectively. is there. FIG. 6 is a graph showing a change in critical micelle concentration (CMC) due to a change in pH in the block copolymer produced according to Example 4. FIG. 7 is a graph showing a change in micelle size due to a change in pH in the block copolymer produced according to Example 4, and is a graph showing a micelle size at pH 8.01. FIG. 8 is a graph showing the change in micelle size due to pH change in the block copolymer produced according to Example 4, and is a graph showing the micelle size at pH 7.42. FIG. 9 is a graph showing the change in micelle size due to pH change in the block copolymer produced according to Example 4, and is a graph showing the micelle size at pH 7.23. FIG. 10 is a graph showing the change in micelle size in the block copolymer produced according to Example 4, and showing the change in micelle size in the pH range of 6.3 to 8.0. FIG. 11 is a graph showing the change over time in the molecular weight of the block copolymers produced according to Example 4 and Example 11, and is a graph showing the molecular weight at pH 7.4. FIG. 12 is a graph showing the change over time in the molecular weight of each of the block copolymers produced according to Examples 11 to 15, and showing the molecular weight at pH 7.4. FIG. 13 is a graph showing the change over time in the molecular weight of each of the block copolymers produced according to Examples 16 to 20, and is a graph showing the molecular weight at pH 7.4.

Claims (22)

(A) a polyethylene glycol-based compound (A);
(B) at least one poly (amino acid) compound selected from the group consisting of poly (β-amino ester) and poly (amidoamine) or a copolymer of the compound (B)
A pH-sensitive block copolymer obtained by copolymerization of   In the block copolymer, at least one poly (amino acid) compound selected from the group consisting of poly (β-amino ester) and poly (amidoamine) or a copolymer of the compound is pH 7.0. The pH-sensitive block copolymer according to claim 1, characterized in that it contains a tertiary amine group that is ionized in the following.   The block copolymer forms micelles in the range of pH 7.2 to 7.4, and the formed micelles collapse in the range of pH 6.5 to 7.0. The pH-sensitive block copolymer as described. The block copolymer has the following general formula (I), (II) or (III):

[Where
R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, wherein x is a natural number in the range of 1 to 10,000,
R ₁ and R ₂ are each an alkyl group having 1 to 20 carbon atoms,
R ₃ is an alkyl group having 1 to 30 carbon atoms,
R ₄ is an alkyl group having 1 to 20 carbon atoms,
y is a natural number in the range of 1 to 10,000]
The pH-sensitive block copolymer according to claim 1, which is a compound represented by the formula:   The pH-sensitive block copolymer according to claim 1, wherein the molecular weight of the block copolymer is in the range of 1000 to 20,000.   The content ratio of the polyethylene glycol (A) -based block and the pH-sensitive block in the block copolymer is 5 to 95% by weight: 5 to 95% by weight. pH-sensitive block copolymer.   The pH-sensitive block copolymer according to claim 1, wherein the polyethylene glycol-based compound includes a functional group selected from the group consisting of acrylate and methacrylate.   The pH-sensitive block copolymer according to claim 1, wherein the polyethylene glycol-based compound has a molecular weight in the range of 500 to 5,000. The poly (amino acid) compound is
(A) a bisacrylate or bisacrylamide compound;
(B) The pH-sensitive block copolymer according to claim 1, which is obtained by polymerizing an amine compound.   The bisacrylate compound is ethylene glycol diacrylate, 1,4-butanediol diacrylate, 1,3-butanediol diacrylate, 1,6-hexanediol diacrylate, 1,6-hexanediol ethoxylate diacrylate, 1 , 6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, 1,7-heptanediol diacrylate, 1,8-octanediol The pH-sensitive block according to claim 9, wherein the block is one or more selected from the group consisting of diacrylate, 1,9-nonanediol diacrylate and 1,10-decanediol diacrylate. Copolymer.   The bisacrylamide-based compound is one or more selected from the group consisting of N, N'-methylenebisacrylamide and N, N'-ethylenebisacrylamide. pH-sensitive block copolymer.   The pH-sensitive block copolymer according to claim 9, wherein the amine-based compound is a primary amine compound or a secondary amine-containing diamine compound.   The primary amine compound is 3-methyl-4- (3-methylphenyl) piperazine, 3-methylpiperazine, 4- (bis) piperazine, 4- (phenylmethyl) piperazine, 4- (1-phenylethyl) piperazine 4- (1,1-dimethoxycarbonyl) piperazine, 4- (2- (bis- (2-propenyl) amino) ethyl) piperazine, methylamine, ethylamine, butylamine, hexylamine, 2-ethylhexylamine, 2-piperidine -1-ethylamine, C-aziridin-1-yl-methylamine, 1- (2-aminoethyl) piperazine, 4- (aminomethyl) piperazine, N-methylethylenediamine, N-ethylethylenediamine, N-hexylethylenediamine, picolamine , And adenine And wherein the or at least one block copolymer of the pH-sensitive of claim 12.   The secondary amine-containing diamine compound is piperazine, piperidine, pyrrolidine, 3,3-dimethylpiperidine, 4,4′-trimethylenedipiperidine, N, N′-dimethylethylenediamine, N, N′-diethylethylenediamine, or imidazo. The pH-sensitive block copolymer according to claim 12, wherein the block copolymer is one or more selected from the group consisting of lysine and diazepine.   The pH-sensitive block copolymer according to claim 9, wherein the molar ratio of the bisacrylate or bisacrylamide compound to the amine compound is in the range of 1: 0.5 to 2.0. . (A) the block copolymer according to any one of claims 1 to 15;
(B) A polymeric micelle type drug composition comprising a physiologically active substance that can be encapsulated in the block copolymer.   The polymer micelle type drug composition according to claim 16, wherein the polymer micelle has a diameter in a range of 10 to 1000 nm.   Use of the block copolymer according to any one of claims 1 to 15 as a carrier for drug transport or disease diagnosis. The following reaction formula 1:

[Where
R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, wherein x is a natural number in the range of 1 to 10,000,
R ₁ is an alkyl group having 1 to 20 carbon atoms,
R ₃ is an alkyl group having 1 to 30 carbon atoms,
y is a natural number in the range of 1 to 10,000]
A method for producing a pH-sensitive block copolymer, which can be obtained by: Reaction formula 2 below:

[Wherein, R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, wherein x is a natural number in the range of 1 to 10,000;
R ₁ and R ₂ are alkyl groups having 1 to 20 carbon atoms,
R ₃ is an alkyl group having 1 to 30 carbon atoms,
y is a natural number in the range of 1 to 10,000]
A method for producing a pH-sensitive block copolymer, which can be obtained by: Reaction formula 3 below:

[Wherein, R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, wherein x is a natural number in the range of 1 to 10,000;
R ₁ and R ₂ are each an alkyl group having 1 to 20 carbon atoms,
R ₄ is an alkyl group having 1 to 20 carbon atoms,
y is a natural number in the range of 1 to 10,000]
A method for producing a pH-sensitive block copolymer, which can be obtained by: (A) one or more compounds selected from the group consisting of a compound containing an ester group and a tertiary amine group, and a compound containing an amide group and a tertiary amine group, or a copolymer of the compound,
(B) A method for producing a pH-sensitive block copolymer capable of forming micelles by copolymerizing a hydrophilic or amphiphilic compound.

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